Ascorbic acid is a publicly known reducing substance as vitamin C, and it is well known that when it is coexistent in a sample to be examined, for instance, a component in a body fluid, a positive or negative measuring error is produced by the reducing property of ascorbic acid in quantitative measurement of an intended component in the sample to be examined by utilizing oxidation-reduction reaction.
Meanwhile, there have been reported as the ascorbic acid decomposing methods (1) a method using ascorbate oxidase (Japanese Patent Publication No. 39,198/1981), (2) a method using iodic acid or a salt thereof, (3) a method using periodic acid or the like or a salt thereof (Japanese Patent Laid-Open No. 109,595/1981; Japanese Patent Laid-Open No. 151,358/1981; Japanese Patent Laid-Open No. 107,161/1981) and so on.
However, the above method (1) using ascorbate oxidase has the defects that since ascorbate oxidase is an enzyme, it has a problem peculiar to the enzymes, that is, a poor thermal stability and a poor storage stability. In addition, although it is strongly demanded to develop a color through one step reaction by using a one liquid type reagent in which necessary reagents are all present in a single liquid or reagents to be gathered in a single liquid in a measuring system in the case of the quantitative measurement of the intended component in the sample to be examined by utilizing the oxidation-reduction reaction, when the above method (1) is applied to this one step reaction, unless a large amount of ascorbate oxidase is used, an oxidase first acts upon a substrate as the intended component in the measurement, so that producing reaction of hydrogen peroxide with the oxidase unfavorably proceeds preferentially when ascorbic acid to be decomposed is still present. There is also a problem that when ascorbate oxidase is used in a large amount to avoid this, it is uneconomical due to its high price.
Further, the method using iodic acid or the salt thereof and the method using periodic acid or the like or the salt thereof have the problem that the enzymatic activity of the oxidase and the like is interfered by the oxidizing action of the oxidizing agent in some case. Moreover, even if the oxidase acts upon the substrate without its enzymatic activity being interfered to quantitatively generate hydrogen peroxide, there has not been developed such an oxidizable coloring reagent up to now that an appropriate pH of coloring of the oxidizable coloring reagent for leading the generated hydrogen peroxide to a coloring system is in coincidence with an appropriate pH at which ascorbic acid is decomposed with iodic acid or the salt thereof or periodic acid or the like or the salt thereof, so that the above method (2) or (3) can not be applied to the one step reaction. Furthermore, since when such periodic acids or the like or the salt thereof is used, a decomposing agent such as an alcohol or an aldehydes is required to decompose an excessive amount of periodic acid or the like or the salt thereof the above method (3) can not be applied to the one step reaction from this respect.
On the other hand, the reaction in which ascorbic acid is oxidized in the presence of divalent copper ion (Cu.sup.2+ ion) to produce dehydroascorbic acid and H.sub.2 O.sub.2 has been formerly known (See E. S. GUZMAN BARRON, R. H. DeMEIO, AND FRIEDRICH KLEMPERER, J. Biol. Chem. 112, 625-640 (1936)). However, this is a reaction in which H.sub.2 O.sub.2 is produced together with dehydroascorbic acid, and in order to apply this decomposition reaction to a reaction in which H.sub.2 O.sub.2 produced by acting an oxidase upon a substrate is measured to quantitatively analyze the intended component in a sample to be examined, the amounts of H.sub.2 O.sub.2 produced in both of these reactions must be able to be discerned and detected, but needless to say, such is substantially impossible.
Therefore, it has not been hit upon at all to try to apply such a reaction that ascorbic acid is decomposed through oxidation in the presence of Cu.sup.2+ ion to the reaction in which H.sub.2 O.sub.2 produced by acting the oxidase upon the substrate is measured to to quantitatively analyze the intended component in the sample to be examined.
It is an object of the present invention to provide a method of decomposing ascorbic acid without producing hydrogen peroxide.
Further, it is another object of the present invention to provide a decomposition method which can effectively remove the effects of ascorbic acid contained in a reaction system in the quantitative measurement of a component in a body fluid in which H.sub.2 O.sub.2 is produced by acting an oxidase upon a substrate, and the thus produced H.sub.2 O.sub.2 is quantitatively measured.
It is still another object of the present invention to provide a method of decomposing ascorbic acid, in which the decomposition of ascorbic acid and a reaction in which a sample to be examined is subjected to enzyme reaction to be led to a coloring system can be carried out by one step.